Currently, magnetic resonance (MR), ultrasonic (US) and computer tomographic (CT) imaging techniques are major tools for clinical diagnosis of diseases and evaluation of therapeutic interventions. Confocal microscopic imaging techniques constitute a state-of-the-art approach to study progression of diseases in ex vivo preparations of tissue and cells of animal models and to evaluate potential treatments, including stem cells, pharmaceuticals and device implants.
Confocal microscopy is an indispensable tool in cell biology because the optical sectioning ability of confocal microscopic imaging enables the study of molecular and morphologic changes in thick biologic specimens with sub-micrometer resolution. Typically, confocal microscopy has not been used to examine living tissue because of the need for close association between microscope instrumentation and the imaged tissue, toxic or expensive fluorescent dyes for image contrast, and relatively long image acquisition times. Despite these challenges, confocal microscopy techniques have been shown to provide valuable diagnostic information for various disease states. Studies with biopsy specimens suggest that confocal imaging can provide useful diagnostic information about the presence of precancerous lesions; confocal images of normal and dysplastic cervical biopsy specimens obtained with a confocal reflectance microscope showed a strong correlation between nuclear morphologic features extracted from confocal images and histopathologic diagnosis.
Confocal microscopic imaging techniques create high resolution images and differs from conventional optical microscopy in that it uses a condenser lens to focus illuminating light of specific wavelengths from a light source, e.g. laser, into a very small, diffraction limited spot within a specimen, and an objective lens to focus the light emitted from that spot onto a small pinhole in an opaque screen. A detector, which is capable of quantifying the intensity of the light that passes through the pinhole at any instant, is located behind the screen. Because only light from within the illuminated spot is properly focused to pass through the pinhole and reach the detector, any stray light from structures above, below, or to the side of the illuminated spot are filtered out. The image resolution is therefore greatly enhanced as compared to other conventional approaches.
In a scanning confocal microscopic imaging system, a coherent image is built up by scanning point by point over the desired field of view and recording the intensity of the light emitted from each spot, as small spots are illuminated at any one time. Scanning can be accomplished in several ways, including for example and without limitation, via laser scanning. Confocal microscopic imaging system are commercially available through entities such as Carl Zeiss, Nikon, and Olympus. An exemplary confocal is described in U.S. Pat. No. 6,522,444 entitled “Integrated Angled-Dual-Axis Confocal Scanning Endoscopes,” which is assigned to Optical Biopsy Technologies Inc.
The ability to obtain confocal images of normal and diseased tissue in vivo is limited by the ability to bring the tissue of interest in close proximity to the microscope objective. Flexible confocal microscopic imaging systems incorporating either a solitary optical fiber or a fiber optic imaging bundle are needed to facilitate in vivo imaging of less accessible organ sites. However, a major obstacle for application of confocal microscopic imaging techniques is related to the introduction of fluorescent dyes into biological tissue. Commonly, introduction of dye is performed by infusion or systemic needle injection. Disadvantages of these methods include, for example, the high dosing requirements, washout and inhomogeneous distribution of the fluorescent dye.